Seed-specific increased expression of 2S albumin promoter of sesame qualifies it as a useful genetic tool for fatty acid metabolic engineering and related transgenic intervention in sesame and other oil seed crops

The sesame 2S albumin (2Salb) promoter was evaluated for its capacity to express the reporter gusA gene encoding β-glucuronidase in transgenic tobacco seeds relative to the soybean fad3C gene promoter element. Results revealed increased expression of gusA gene in tobacco seed tissue when driven by sesame 2S albumin promoter. Prediction based deletion analysis of both the promoter elements confirmed the necessary cis-acting regulatory elements as well as the minimal promoter element for optimal expression in each case. The results also revealed that cis-regulatory elements might have been responsible for high level expression as well as spatio-temporal regulation of the sesame 2S albumin promoter. Transgenic over-expression of a fatty acid desaturase (fad3C) gene of soybean driven by 2S albumin promoter resulted in seed-specific enhanced level of α-linolenic acid in sesame. The present study, for the first time helped to identify that the sesame 2S albumin promoter is a promising endogenous genetic element in genetic engineering approaches requiring spatio-temporal regulation of gene(s) of interest in sesame and can also be useful as a heterologous genetic element in other important oil seed crop plants in general for which seed oil is the harvested product. The study also established the feasibility of fatty acid metabolic engineering strategy undertaken to improve quality of edible seed oil in sesame using the 2S albumin promoter as regulatory element.     

Isolation and Characterization of Fruit-specific Promoters ACS4 and EXP1 from Tomato (Solanum lycopersicum L)

Promoter engineering in plants holds a great promise for understanding complexity of genetic regulatory system in response to specific internal and external cues and for crop improvement. In the present investigation, we report characterization of two fruit-specific promoters SIACS4 and SIEXP1 that were isolated from tomato (Solanum lycopersicum L cv Pusa Ruby). In silico analysis of the cloned promoter sequences revealed the presence of a seed-specific cis-element in SIACS4 and several putative seed, embryo and endosperm-specific cis-elements in SIEXP1 in addition to fruit-specific ethylene responsive regulatory elements. The fruit- and seed-specific expression of both the promoters was analyzed in transgenic tomato lines expressing the promoter:: GUS fusion constructs. The SIACS4 promoter (?1 to ?373) showed GUS activity restricted specifically to flower buds and seeds in fruits. On the contrary, the SIEXP1 promoter (?1 to ?769) showed high level of expression in seeds as compared to fruit tissues at different stages of fruit ripening. No GUS expression was observed in leaves satisfying the fruit-specific nature of both the promoters. Based on deletion analysis, minimal promoters SIACS4DL2 (?1 to ?126) and SIEXP1DL1 (?1 to ?254) were identified which can be used to drive tissue-specific expression of transgenes for introducing traits of agronomic importance such as resistance to fruit borer and for enhancing both nutritional and keeping quality of tomato fruits.     

In Silico and Deletion Analysis of Upstream Promoter Fragment of S-Adenosyl Homocysteine Hydrolase (SAHH1) Gene of Arabidopsis Leads to the Identification of a Fragment Capable of Driving Gene Expression in Developing Seeds and Anthers

S-adenosyl homocysteine hydrolase (SAHH) is a key enzyme in methylation metabolism of eukaryotes. A 1585 by fragment upstream to ATG of SAHH1 gene, was fused with a promoter-less β-Glucuronidase (GUS) gene and mobilized into Arabidopsis by Agrobacterium-mediated floral transformation to generate transgenic Arabidopsis. This fragment was found to drive constitutive expression of GUS in T₂ progeny of transgenic Arabidopsis. In silico analysis of the promoter region of SAHH1 suggested the presence of several cis-regulatory motifs including seed-specific motifs as well as anther-specific motifs in the 376 by (upstream to TSS of SAHH1) promoter fragment. Based on the partial deletion analysis carried out in the promoter region of SAHH1 (At4gl3940) this 376 by promoter fragment was found to be capable of driving GUS expression in developing seeds and in some anthers/micros pores.     

Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5 control region of the napA gene were fused to the reporter gene -glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between –1101 and –309 resulted in increased GUS activity. In contrast, deletion of sequences between –309 and –211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between –152 and +44. Further 5 deletion of the fragment to –126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between –148 to –120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to –152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.     

Screening and identification of soybean seed-specific genes by using integrated bioinformatics of digital differential display,microarray, and RNA-seq data

Soybean is one of the most economically important crops in the world. Soybean seeds have abundant protein and lipid content and very high economic value. In this study, a total of 184 seed-specific genes were obtained using online microarray databases, DDD, and RNA-seq data. The reported seed-specific genes in soybean and the 184 seed-specific genes analyzed in this paper were compared. Of the screened genes, 26 were common to both previous reports and the current screening. Meanwhile, 90 of the 184 genes have homologous counterparts in Arabidopsis, among which 24 have seed-specific expression, as indicated by microarray data for Arabidopsis. Furthermore, promoter analysis showed that almost all seed-specific genes contain at least one seed specific-related element. Seed-specific element Skn-1 motif exists in most, if not all, of the seed-specific genes screened. Five genes were randomly selected from 184 soybean seed specific gene pool and their expressions were quantified using quantitative real time polymerase chain reaction (qRT-PCR) to further confirm the specificity of the screened genes. The results indicated that all five genes showed seed-specific expression. Moreover, the identification of genes with seed-specific expression screened in this study provides information valuable to the in-depth study of soybean.     

Molecular basis of seed lipoxygenase null traits in soybean line OX948

The poor stability and off-flavors of soybean oil and protein products can be reduced by eliminating lipoxygenases from soybean seed. Mature seeds of OX948, a lipoxygenase triple null mutant line, do not contain lipoxygenase proteins. The objective of this study was to determine the molecular basis of the seed lipoxygenase null traits in OX948. Comparisons of the sequences for lipoxygenase 1 (Lx1) and lipoxygenase 2 (Lx2) genes in the mutant (OX948) with those in a line with normal lipoxygenase levels (RG10) showed that the mutations in these genes affected a highly conserved group of six histidines necessary for enzymatic activity. The OX948 mutation in Lx1 is a 74?bp deletion in exon 8, which introduces a stop codon that prematurely terminates translation. A single T?CA substitution in Lx2 changes histidine H532 (one of the iron-binding ligands essential for L-2 activity) to glutamine. The mutation in the lipoxygenase 3 (Lx3) gene in OX948 is in the promoter region and represents two single base substitutions in a cis-acting AAATAC paired box. All three mutations would result in the loss of lipoxygenase activity in mature seed. The seed lipoxygenase gene mutation-based molecular markers could be used to accelerate and simplify breeding efforts for soybean cultivars with improved flavor.     

Cotton α-Globulin Promoter: Isolation and Functional Characterization in Transgenic Cotton,Arabidopsis, and Tobacco

Globulins are the most abundant seed storage proteins in cotton and, therefore, their regulatory sequences could potentially provide a good source of seed-specific promoters. We isolated the putative promoter region of cotton -globulin B gene by gene walking using the primers designed from a cotton staged embryo cDNA clone. PCR amplified fragment of 1108 bp upstream sequences was fused to gusA gene in the binary vector pBI101.3 to create the test construct. This was used to study the expression pattern of the putative promoter region in transgenic cotton, Arabidopsis, and tobacco. Histochemical GUS analysis revealed that the promoter began to express during the torpedo stage of seed development in tobacco and Arabidopsis, and during cotyledon expansion stage in cotton. The activity quickly increased until embryo maturation in all three species. Fluorometric GUS analysis showed that the promoter expression started at 12 and 15 dpa in tobacco and cotton, respectively, and increased through seed maturation. The strength of the promoter expression, as reflected by average GUS activity in the seeds from primary transgenic plants, was vastly different amongst the three species tested. In Arabidopsis, the activity was 16.7% and in tobacco it was less than 1% of the levels detected in cotton seeds. In germinating seedlings of tobacco and Arabidopsis, GUS activity diminished until it was completely absent 10 days post imbibition. In addition, absence of detectable level of GUS expression in stem, leaf, root, pollen, and floral bud of transgenic cotton confirmed that the promoter is highly seed-specific. Analysis of GUS activity at individual seed level in cotton showed a gene dose effect reflecting their homozygous or hemizygous status. Our results show that this promoter is highly tissue-specific and it can be used to control transgene expression in dicot seeds.     

Cloning of a new LEA1 gene promoter from soybean and functional analysis in transgenic tobacco

LEA1 gene from Glycine max can be expressed in late-embryo stage of plants, and respond to salinity and dehydration stress. To elucidate the mechanism for stress tolerance and high expression in seeds, we isolated and characterized the promoter of LEA1 gene (EQ, 1997 bp) starting the 5′LEA1 coding region. A deletion mutant of EQ promoter (ED) and the full length promoter (EQ) were fused to GUS reporter gene and transformed into the tobacco leaf discs. The results indicated that expression of the reporter gene (GUS) could be regulated by EQ promoter, and was stronger than the mutant under the stress conditions. Also, the expression level of GUS gene driven by EQ promoter in transgenic tobacco seeds was significantly higher than that by the mutant promoter, which meant that it had a better tissue-specificity. Therefore, the active domain for the promoter was located between ?1997 and ?1000 bp. Additionally, the activity of EQ promoter was 2.1-, 3.3- and 0.4- times stronger than the activity of promoter CaMV35S under salt (24 h), drought (10 h) or ABA (24 h), respectively. Meanwhile, the GUS activity of EQ promoter in seeds was 1.8-fold stronger compared to the promoter CaMV35S. In summary, the new promoter (EQ) is bi-functional, stress-inducible and seed-specific. These findings provide a further understanding for the regulation of LEA1gene expression, and suggest a new way for improving seed quality under saline and alkaline land.     

The promoter of the wheat puroindoline-a gene (<Emphasis Type="Italic">PinA</Emphasis>) exhibits a more complex pattern of activity than that of the <Emphasis Type="Italic">PinB</Emphasis> gene and is induced by wounding and pathogen attack in rice

Puroindolines form the molecular basis of wheat grain hardness. However, little is known about puroindoline gene regulation. We previously reported that the Triticum aestivum puroindoline-b gene (PinB) promoter directs β-glucuronidase gene (uidA) seed-specific expression in transgenic rice. In this study, we isolated a puroindoline-a gene (PinA), analyzed PinA promoter activity by 5′ deletions and compared PinA and PinB promoters in transgenic rice. Seeds of PinA-1214 and PinB-1063 transgenic plants strongly expressed uidA in endosperm, in the aleurone layer and in epidermis cells in a developmentally regulated manner. The GUS activity was also observed in PinA-1214 embryos. Whereas the PinB promoter is seed specific, the PinA promoter also directed, but to a lower level, uidA expression in roots of seedlings and in the vascular tissues of palea and pollen grains of dehiscent anthers during flower development. In addition, the PinA promoter was induced by wounding and by Magnaporthe grisea. By deletion analysis, we showed that the “390-bp” PinA promoter drives the same expression pattern as the “1214-bp” promoter. Moreover, the “214-bp” PinA promoter drives uidA expression solely in pollen grains of dehiscent anthers. The presence of putative cis-regulatory elements that may be related to PinA expression is discussed from an evolutionary point of view. By electrophoretic mobility shift assay, we showed that putative cis-elements (WUN-box, TCA motifs and as-1-like binding sites) whose presence in the PinA promoter may be related to wounding and/or the pathogen response form complexes with nuclear extracts isolated from wounded wheat leaves.